The electcrochemistry of oxidoreductases has received considerable attention in relation to applications in enzyme electrodes. See, e.g., A. P. F. Turner et al., Biosensors 1:85-115 (1985); F. W. Scheller et al., Biosensors 1:135-160 (1985); M. Mascini et al., Biosensors 2:147-172 (1985); and A. P. F. Turner and M. F. Cardosi, in Biosensors: Fundamentals and Applications, at pp. 257-275 (Eds. A. P. F. Turner, I. Karube and G. S. Wilson), Oxford: Oxford University Press, 1987. Many of the same considerations apply to their use in immunoassay and other enzyme-labelled assays e.g. DNA and RNA probe assays. In particular, highly efficient coupling of enzymic activity to the electrochemical detector is essential for sensitive and rapid assays. A number of approaches for the realisation of electron transfer from biological systems to amperometric electrodes have been described, but arguably the most effective is the use of low molecular weight mediators to shuttle electrons between the catalyst and an electrode. Various mediators that have been reported for use in enzyme electrodes, such as ferricyanide (P. Racine et al., Experientia 18:525-534 (1971)), tetracyano-p-quinodimethane (J. J. Kulys et al., Biochim. Biophys. Acta. 744:57-63 (1983)) and ferrocene (W. J. Aston et al., in Charge and Field Effects in Biosystems, at pp. 491-498 (Eds. M. J. Allen and P. N. R. Underwood), Tunbridge Wells, England: Abacus Press, 1984; A. E. G. Cass et al., Anal. Chem. 56:667-671 (1984); and A. P. F. Turner et al., Anal. Chim. Acta. 163:161-174 (1984) could also be useful in immunosensors. ##STR2## Mediated enzyme-linked immunoassay, in which a GOD label was monitored using a ferrocene derivative, was first reported in 1985 by G. A. Robinson et al. in Clin. Chem. 31:1449-1452 (1985). A more elegant possibility is the use of the mediator molecule as a label. Weber et al. (Analyt. Lett. 12:1-9 (1979)) produced a conjugate of morphine and ferrocene carboxylic acid. They showed that the electrochemical oxidation of the ferrocene label was reduced when morphine antibody bound the conjugate and used this principle in a displacement assay for codeine (see (a) below). Since the key to practical oxidoreductase electrochemistry is the availability of a mediator such as ferrocene, it was apparent that this principle could be used to trigger an electrochemically coupled enzyme-catalysed reaction (see (b) below). ##STR3## The effective recycling of the ferrocene by GOD results in a further amplification of the signal over electrochemical noise due to electroactive substances present in the sample.
Electrochemically coupled enzyme reactions may also be activated by providing missing cofactors or coenzymes, as described by T. T. Ngo et al., Appl. Biochem. Biotechnol. 11:663-670 (1985). Quinoprotein dehydrogenases could prove particularly valuable in this respect.
An immunoassay for prostatic acid phosphatase (PAP), a prostate tumor marker from human serum, which relies on enzyme amplification is shown below. See M. F. Cardosi et al., "An Electrochemical Immunoassay Using Enzyme Amplification", in Second International Meeting on Chemical Sensors (Eds. J. L. Acoutrier et al.), 1985. ##STR4## The catalytic activity of the enzyme label (alkaline phosphatase) used in a sandwich assay is monitored by the addition of the substrate NADP.sup.+ leading to the formation of the dephosphorylated product NAD.sup.+. The NAD.sup.+ formed enters a redox cycle involving the enzymes alcohol dehydrogenase and diaphorase leading to the reduction of a mediator (ferricyanide). Electrons from the NAD.sup.+ /NADH redox cycle passed via the diaphorase to the Fe.sub.III (CN).sub.6 /Fe.sub.II (CN).sub.6 couple. The reduced species FE.sub.II (CN).sub.6 was reoxidised at a platinum electrode at 450 mV versus a saturated calomel electrode producing an amperometric response.
Similar principles may be applied to other affinity reactions such as DNA and RNA probe assays.
Amperometric enzyme electrodes have been investigated in which the electrode has a conductive surface comprising an organic solid with metal-like electrical conductivity ("organic metal"). These substances are formed as charge-transfer complexes between an electron donor molecule and an electron acceptor molecule. The principal investigations have been with 7,7,8,8-tetracyanoquinodimethane (TCNQ) as electron acceptor and N-methyl-phenazinium (NMP) as electron donor, but the possibility of TTF.sup.+ TCNQ.sup.- complexes has also been considered. See J. J. Kulys, Biosensors 2:3-13 (1986). However, the present invention is dealing with the use of TTF in a different context; uncomplexed, as a mediator of electron transfer.